Glucose-transport related genes, polypeptides, and methods of use thereof

ABSTRACT

Methods and compositions for modulating glucose transport are provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application No. 60/632,462, filed Dec. 2, 2004, the contents ofwhich are hereby incorporated by reference in their entirety.

The work described herein was funded, in part, through grants from theNational Institutes of Health (Grant Nos. DK30898 and DK60837-03). TheUnited States government may, therefore, have certain rights in theinvention.

TECHNICAL FIELD

This invention relates to molecular biology, cell biology, glucosetransport, and diabetes.

BACKGROUND

Insulin stimulates glucose transport in muscle and fat. One of the mostcritical pathways that insulin activates is the rapid uptake of glucosefrom the circulation in both muscle and adipose tissue. Most ofinsulin's effect on glucose uptake in these tissues is dependent on theinsulin-sensitive glucose transporter, GLUT4 (reviewed in Czech andCorvera, 1999, J. Biol. Chem., 274:1865-1868, Martin et al., 1999, CellBiochem. Biophys., 30:89-113, Elmendorf et al., 1999 Exp. Cell Res.,253:55-62). The mechanism of insulin action is impaired in diabetes,leading to less glucose transport into muscle and fat. This is thoughtto be a primary defect in type II diabetes. Potentiating insulin actionhas a beneficial effect on type II diabetes. This is believed to be themechanism of action of the drug Rezulin (troglitazone).

Type II diabetes mellitus (non-insulin-dependent diabetes) is a group ofdisorders, characterized by hyperglycemia that can involve an impairedinsulin secretory response to glucose and insulin resistance. One effectobserved in type II diabetes is a decreased effectiveness of insulin instimulating glucose uptake by skeletal muscle. Type II diabetes accountsfor about 85-90% of all diabetes cases. In some cases of type IIdiabetes the underlying physiological defect appears to bemultifactoral.

SUMMARY

The invention is based, at least in part, on the discovery of geneproducts that regulate glucose transport in cells. The genes and geneproducts described herein are novel targets for modulation for thetreatment of disorders in which glucose metabolism is disregulated, suchas diabetes.

Accordingly, in one aspect, the invention features methods foridentifying a candidate agent that modulates expression or activity of aglucose transport-related polypeptide. The methods include, for example:(a) providing a sample including a glucose-transport related polypeptideor a nucleic acid encoding the polypeptide, wherein theglucose-transport related polypeptide is a gene product of a gene inTable 1 or Table 2, or a homolog thereof (e.g., a human homolog); (b)contacting the sample with a test compound; (c) evaluating expression oractivity of the glucose transport-related polypeptide in the sample; and(d) comparing the expression or activity of the glucosetransport-related polypeptide of (c) to expression or activity of theglucose transport-related polypeptide in a control sample lacking thetest compound, wherein a change in glucose transport-related polypeptideexpression or activity indicates that the test compound is a candidateagent that can modulate the expression or activity of the glucosetransport-related polypeptide.

In various embodiments, the glucose transport-related polypeptide is agene product of a gene in Table 1, e.g., Peroxin 13 (Pex 13),ADP-ribosylation factor-like 6 interacting protein (Ar16ip2), Superoxidedismutase 1 (SOD), a product of Fat specific gene 27 (FSP27), Frizzledhomolog 4 (Fzd4), Synaphin 3 (Sycp3), Protein tyrosine phosphatasereceptor type A (Ptpra), Conserved helix-loop-helix ubiquitous kinase(Chuk, or IKKα), Mitogen-activated protein kinase kinase kinase kinase 4(Map4k4), PCTAIRE-motif protein kinase 1 (Pctk1), PCTAIRE-motif proteinkinase 3 (Pctk3), PFTAIRE protein kinase 1 (Pftk1), Mitogen activatedprotein kinase 8 (JNK1), Mitogen activated protein kinase 9 (JNK2),MAP/microtubule affinity-regulating kinase 3 (Mark3), Interleukin-1receptor-associated kinase 1 (IRAK1), or Expressed in non-metastaticcells 3 (Nme3). In various embodiments, the glucose transport-relatedpolypeptide includes an amino acid sequence at least 50, 60, 70, 80, 90,95, 96, 99, or 100% identical to the gene product of a gene in Table 1.The polypeptide can be a human polypeptide (e.g., a human polypeptideencoded by a gene in Table 1 or a human homolog of a gene in Table 1).

In various embodiments, the glucose transport-related polypeptide is agene product of a gene in Table 2, e.g., a gene product of one of thefollowing clones from The Institute of Physical and Chemical Research(RIKEN): 9130022E05Rik, 2900045N06Rik, 4930402E16Rik, 5730403B10Rik,F830029L24Rik, G430055L02Rik, or a human homolog thereof; or the geneproduct is ATP-binding cassette, sub family F (Abcf1), Cysteine-richmotor neuron 1 (Crim1), Dishevelled segment polarity protein homolog(Dvl1), NAD(P) dependent steroid dehydrogenase-like protein (Nsdhl),Integrin linked kinase (Ilk), Par-6 partitioning defective 6 homologgamma (Pard6g), or Lipin (Lpin1); or the gene product of D11Ertd498 orD19Ertd7O3e. In various embodiments, the glucose transport-relatedpolypeptide includes an amino acid sequence at least 50, 60, 70, 80, 90,95, 96, 99, or 100% identical to the gene product of a gene in Table 2.

The sample used in the methods can be or include a cell (e.g., anadipocyte) or can be a cell-free sample. The expression or activity ofthe glucose transport-related polypeptide can be evaluated, e.g., usinga cell-free or cell-based assay. Modulation of expression can beevaluated using an antibody. In one embodiment, the evaluating includesdetermining whether glucose transport is modulated in the presence ofthe test compound, e.g., by determining glucose uptake.

The test compound evaluated in the method can be a polynucleotide, apolypeptide, a small non-nucleic acid organic molecule, a smallinorganic molecule, or an antibody. For example, the test compound canbe an antisense oligonucleotide, an inhibitory RNA, or a ribozyme.

Glucose transport may be increased or decreased in the presence of thetest compound.

In various embodiments, the glucose transport-related polypeptide is akinase. In such embodiments, the evaluating can include determiningphosphorylation of a substrate by the kinase, e.g., using a kinaseassay.

The methods can include steps in which the effect of the test compoundon expression or activity of the glucose transport-related polypeptideis evaluated in vivo, e.g., using an animal model, such as an animalmodel of diabetes.

In another aspect, the invention features methods for modulating glucosetransport in a cell. These methods include, for example; providing acell; contacting the cell (e.g., in vitro or in vivo) with an agent thatmodulates expression or activity of a glucose transport-relatedpolypeptide, thereby modulating glucose transport in the cell.

The test compound that modulates expression or activity of a glucosetransport-related polypeptide can be an agent identified by a methoddescribed herein, e.g., a method including the following steps: (a)providing a sample including the glucose-transport related polypeptideor a nucleic acid encoding the polypeptide; (b) contacting the samplewith a test compound; (c) evaluating expression or activity of theglucose transport-related polypeptide in the sample; and (d) comparingthe expression or activity of the glucose transport-related polypeptideof (c) to expression or activity of the glucose transport-relatedpolypeptide in a control sample lacking the test compound, wherein achange in glucose transport-related polypeptide expression or activityindicates that the test compound is a candidate agent that can modulatethe expression or activity of the glucose transport-related polypeptide.

The test compound employed in the method of modulating glucose transportin a cell can modulate the expression or activity of a gene product of agene in Table 1 or Table 2. The test compound may decrease or increaseexpression or activity of a gene product of a gene in Table 1. The testcompound can be a polynucleotide, a polypeptide, a small non-nucleicacid organic molecule, a small inorganic molecule, and an antibody. Forexample, the test compound is a small inhibitory RNA. The test compoundcan be selected from the group consisting of an antisenseoligonucleotide, an inhibitory RNA, or a ribozyme.

The methods for modulating glucose transport in a cell can furtherinclude contacting the cell with a second agent that modulatesexpression or activity of a glucose transport-related polypeptide.

The invention also features methods for increasing insulin-stimulatedglucose uptake in a subject. The methods include: administering to thesubject an agent that decreases expression or activity of a gene productof a gene in Table 1 in an amount sufficient to modulate glucosemetabolism in a cell of the subject, thereby increasinginsulin-stimulated glucose uptake in the subject. For example, thesubject can be at risk for or suffering from a disorder or conditionrelated to glucose metabolism such as type I diabetes, type II diabetes,or obesity.

The invention also provides methods for modulating glucose metabolism ina subject by administering to the subject an agent that increasesexpression or activity of a gene product of a gene in Table 2 in anamount sufficient to modulate glucose metabolism in a cell of thesubject, thereby modulating glucose metabolism in the subject.

Also featured herein are compositions that include a nucleic acidencoding an inhibitory RNA that targets an RNA encoded by a gene ofTable 1. In one embodiment, the inhibitory RNA is a small inhibitoryRNA.

The invention further provides compositions that include an antisensenucleic acid that inhibits the function of a gene product of a gene ofTable 1.

The invention also features methods for diagnosing a disorder orcondition related to glucose metabolism by evaluating the expression oractivity of one or more gene products of the genes in Tables 1 and 2.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. All cited patents, patent applications, and references(including references to public sequence database entries) areincorporated by reference in their entireties for all purposes. U.S.Provisional App. No. 60/632,462, filed Dec. 2, 2004, is incorporated byreference in its entirety for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of graphs depicting the levels and dose dependence ofinsulin-stimulated deoxyglucose uptake in cells transfected with siRNAs.Cells were transfected with scrambled (6 nmol), Akt1+Akt2, (4+6 nmol),or PTEN (6 nmol) as controls, and indicated SMARTpool® (6 nmol) siRNAs,stimulated with insulin, and assayed by determining counts per minute(CPMs) of radioactive deoxyglucose uptake in cells treated with controlssiRNA or indicated SMARTpool® siRNAs. The names of each gene productidentified by the screen are listed on the X axes. Protein kinase hits,indicated by the letters arrows and asterisks, were identified usingthis strategy. The data represents the average of 3 independentscreening experiments.

FIG. 2 is a graph depicting the levels and dose dependence ofinsulin-stimulated deoxyglucose uptake in cells transfected with siRNAsidentified in the experiments shown in FIG. 1. Cells were electroporatedwith controls siRNA or each SMARTpool® (6 nmol) siRNA indicated andassayed as described for FIG. 1. Both positive and negative regulatorsof glucose transport were examined. The data represents the average of 3independent screening experiments.

FIG. 3 is graph depicting the effects of RNAi-based silencing ofspecific genes on insulin-induced Akt phosphorylation. Culturedadipocytes were transfected with scrambled, PTEN, or the indicatedSMARTpool® siRNAs by electroporation, reseeded for 72 hours, andserum-starved overnight before treatment with insulin for 30 minutes.Cell lysates were resolved by SDS-PAGE and phospho-(Ser⁴⁷³)Akt wasdetected by Western blotting. Quantitations of phosphorylated Akt areplotted. PTEN and IKKβ enhance insulin-stimulated Akt phosphorylation,as indicated by asterisks. Data are representative of three independentexperiments.

FIG. 4 is graph depicting the effects of siRNA on GLUT4 proteinexpression. Cultured adipocytes were transfected with scrambled, or theindicated IKKα, IKKβ, Map4K4, or ILK SMART pool® siRNAs byelectroporation, then reseeded and incubated for 72 hours. Cells werelysed and lysates were resolved by SDS-PAGE. Blots were probed withanti-GLUT4 antibody. IKKα and Map4K4, but not IKKβ siRNA enhance GLUT4protein, as indicated by asterisks. ILK siRNA caused decreased GLUT4expression (also indicated by an asterisk). Data are representative ofthree independent experiments.

FIG. 5 is a graph depicting the levels and dose dependence ofinsulin-stimulated deoxyglucose uptake in cells transfected with siRNAsagainst MAPK family members. Cultured adipocytes were transfected withsiRNA pools directed against each of the 22 MAPK family membersexpressed in adipocytes and deoxyglucose uptake by the cells wasdetermined. Arrows indicate siRNAs that had an effect on deoxyglucoseuptake (PTEN and Map4K4).

FIG. 6 is a graph depicting the change in triglyceride levels inadipocytes transfected with either scrambled siRNA or Map4K4 siRNA. Dataare representative of three independent experiments.

FIGS. 7A and 7B are graphs depicting changes in PPARγ, C/EBPα, andMap4K4 mRNA levels in adipocytes as determined by real time PCR analysisof total RNA isolated during differentiation over 11 days. Data are theaverages of two independent experiments and are shown as fold changesover day 0.

FIG. 8 is a graph depicting changes in Map4K4 levels in adipocytestransfected with scrambled, Map4K4, PPARγ, or Map4K4+PPARγ siRNAs.Adipocytes were transfected with siRNA and replated for 72 hours. RNAwas extracted and relative abundance of mRNAs were evaluated by realtime PCR analysis. Data represent the average of four independentexperiments.

FIGS. 9A and 9B are graphs depicting changes in Map4K4 and GLUT4 levelsin adipocytes incubated in the presence or absence of 10 ng/ml TNFα for24 hours. Adipocytes were electroporated with scrambled or Map4K4 siRNAprior to TNFα treatment. Map4K4 and GLUT4 expression were measured byreal time PCR analysis. Data are presented as fold changes over thescrambled siRNA condition and represent the average of three independentexperiments.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

We have identified genes, the polypeptide products of which regulateglucose transport in adipocytes in response to insulin signals. Here, weprovide methods and compositions for modulating expression of thesepolypeptides, and for identifying agents that modulate their expression.The genes encoding the polypeptides we have identified are listed inTables 1 and 2, below. A first subset of the genes we have identifiedencodes polypeptides that are negative regulators of glucose transport.A second subset encodes positive regulators of glucose transport.Decreasing the expression or activity of negative regulators of glucosetransport (e.g., via inhibition of gene expression with antisense orsiRNA or with agents that inhibit the activity of the gene product) canresult in increase glucose uptake, thereby lowering blood glucoselevels. Increasing expression or activity of positive regulators (e.g.,via overexpression of the gene product or via use of agents thatincrease the activity of the regulators) can also enhance insulinaction, thereby promoting glucose uptake.

Negative Regulators of Glucose Transport

We have found that inhibiting expression of the genes listed in Table 1potentiates insulin action by increasing insulin-stimulated glucoseuptake. Potentiation of insulin action is beneficial, e.g., incontrolling blood glucose action in vivo, e.g., in diabetic patients.Thus, inhibiting the expression or activity of these gene products canbe beneficial in the treatment of conditions in which insulin activityor glucose transport is disregulated. TABLE 1 Negative Regulators ofGlucose Transport Gene or Gene Product Name Gene or Gene GenBank ®(abbreviated) Product Name Accession No.* Comments Pex13 Peroxin 13BC023683 Component of peroxisomal translocation machinery Ar16ip2ADP-ribosylation NM_178050 factor-like 6 interacting protein SOD1Superoxide BC057074 Synonym: dismutase 1 B430204E11Rik FSP27 Fatspecific gene Mouse: 27 NM_178373 Human: AY364640 Fzd4 Frizzled homologMouse: 4 NM_008055 Human: AB032417 Sycp3 Synaphin 3 Mouse: NM_011517Human: NM_153694 Ptpra Protein tyrosine M36033 Phosphatase phosphatase(NM_008980) receptor type A Chuk (IKKα) Conserved helix- Mouse: Kinaseloop-helix NM_007700 ubiquitous kinase Human: NM_001278 Map4k4Mitogen-activated Mouse: Kinase protein kinase NM_008696 kinase kinaseHuman: kinase 4 NM_145686 Pctk1 PCTAIRE-motif Mouse: Kinase proteinkinase 1 NM_011049 Human: X66363 Pctk3 PCTAIRE-motif Mouse: Kinaseprotein kinase 3 NM_008795 Human: NM_212502 Pftk1 PFTAIRE proteinNM_011074 Kinase kinase 1 Mapk8-A (JNK1) Mitogen activated NM_016700Kinase protein kinase 8 Mapk9 (JNK2) Mitogen activated NM_016961 Kinaseprotein kinase 9 Mark3 MAP/microtubule NM_022801 Kinaseaffinity-regulating kinase 3 IRAK1 Interleukin-1 NM_008363 Kinasereceptor- associated kinase 1 Nme3 Expressed in non- NM_019730 Kinasemetastatic cells 3*GenBank ® entries in this table refer to murine sequences unlessotherwise noted.

Peroxin-13

Peroxin-13 (Pex13) is a component of the peroxisomal translocationmachinery along with Peroxin-14 and Peroxin-17. Both N- and C-termini ofPex13 are oriented to the cytosol. Pex13 encodes an SH3-containingperoxisomal membrane protein required for the import of proteins intoperoxisomes such as peroxin-14. In humans, mutations in PEX13 candisrupt peroxisome biogenesis and lead to peroxisomal metabolicdysfunction and neurodegenerative disease. Murine PEX13 spans 18 kb andconsists of four exons. Pex13 transcripts were detected in all mousetissues tested, with highest levels in liver and testis. The PEX13 openreading frame predicts a 44.5-kDa protein that displays 91% sequenceidentity to the human protein.

Fat Specific Gene 27

Fat Specific Gene 27 (FSP 27) encodes a protein of 27 kDa (Danesch etal., J Biol. Chem., 267(10):7185-93, 1992). FSP27 encodes a domain thatis found in caspase-activated (CAD) nuclease, which induces DNAfragmentation and chromatin condensation during apoptosis; and in thecell death activator proteins CIDE-A and CIDE-B, which are inhibitors ofCAD nuclease. The two proteins interact through this domain (Enari etal., Nature, 391:43-50, 1998; Sakahira et al., Nature, 391:96-99, 1998).

A human homolog of mouse FSP27, CIDE-3 (cell-death-inducing DFF45-likeeffectors) has been identified (Liang et al., Biochem J.,370:195-203,2003). The nucleic acid sequence of CIDE-3 is found underGenBank® Acc. No. AY364640.

Frizzled Homolog 4

Members of the ‘frizzled’ (FZ) gene family encode 7-transmembrane domainproteins that are receptors for Wnt signaling proteins. The human FZD4gene encodes a deduced 537-amino acid protein that has a cysteine-richdomain (CRD) in the N-terminal extracellular region, 2 cysteine residuesin the second and third extracellular loops, 2 extracellular N-linkedglycosylation sites, and the S/T-X-V motif in the C terminus (Kirikoshiet al., Biophys. Res. Commun., 264: 955-961, 1999; see also GenBank®Acc. No. AB032417). Mutations in FZD4 have been linked with familialexudative vitreoretinopathy (FEVR), a hereditary ocular disordercharacterized by a failure of peripheral retinal vascularization(Robitaille et al., Nat Genet., 32(2):326-30, 2002).

Synaphin 3

Synaphin 3 (also known as Synaptonemal Complex Protein 3, or Sycp3)encodes a protein component of the synaptonemal complex. Humansheterozygous for a mutation in SYCP3 are azoospermic, indicating thatthe Sycp3 is essential for meiotic function in human spermatogenesis(Miyamoto et al., Lancet, 362(9397):1714-9, 2003).

Protein Tyrosine Phosphatase Receptor Type A

Protein Tyrosine Phosphatase Receptor Type A (Ptpra) is a tyrosinespecific protein phosphatase. Although Ptpra has been implicated ininsulin signaling in cultured cells, Ptpra-null mice reveal that thepolypeptide is not essential for mediating the physiological action ofinsulin (Le et al., Biochem. Biophys. Res. Commun., 314 (2):321-329,2004).

Conserved Helix-Loop-Helix Ubiquitous Kinase

Conserved Helix-Loop-Helix Ubiquitous Kinase (Chuk; also known as IKappa B Kinase α, or IKKα) is a serine/threonine kinase that isexpressed in a broad array of tissues and exhibits a high degree ofconservation across species. Chuk contains kinase, leucine zipper, andhelix-loop-helix domains (Mercurio et al., Science, 278(5339):860-6,1997). Phosphorylation of serine residues on the IKB proteins by kinasessuch as IKKα marks them for destruction via the ubiquitination pathway,thereby allowing activation of the NF-kappa-B complex.

Mitogen-activated Protein Kinase Kinase Kinase Kinase 4

Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (Map4k4; alsoknown as NCK-interacting Kinase, or NIK; also referred to herein asMap4k4/NIK) is a serine/threonine kinase that regulates diversesignaling pathways and is essential for mammalian development (Xue etal., Development, 128(9):1559-1572, 2001). This kinase has been shown tospecifically activate MAPK8/JNK. The activation of MAPK8 by Map4k4 isfound to be inhibited by the dominant-negative mutants of MAP3K7/TAK1,MAP2K4/MKK4, and MAP2K7/MKK7, which suggests that this kinase mayfunction through the MAP3K7-MAP2K4-MAP2K7 kinase cascade, and mediatethe TNFα signaling pathway.

Exemplary nucleic acid and amino acid sequences for human Map4k4 arefound under GenBank Nos. NM_(—)145686 and NP_(—)663719, respectively.The N-terminus of the human Map4k4 polypeptide has a catalytic kinasedomain with 11 kinase subdomains (Yao et al., J. Biol. Chem., 274:2118-2125, 1999). Map4k4 shares 47% and 48% amino acid sequence identityto the catalytic domain of Hematopoietic Progenitor Kinase 1 (HPK1) andGerminal Center Kinase, GCK, respectively. Other polypeptides which havebeen shown to interact with human Map4k4 include: Caspase 8, Dockingprotein 1; guanylate binding protein 3; Integrin beta 1; Nck adaptorprotein 1; Solute carrier family 9, isoform A1; RasGAP; solute carrierfamily 9 (sodium/hydrogen exchanger), member 1; and MEKK1.

PCTAIRE-1

PCTAIRE-1 (Pctk1) is a cyclin dependent kinase-related protein found interminally differentiated cells in brain and testis. (Graeser et al., J.Cell Sci., 115:3479-3490, 2002). Like cyclin dependent kinases, Pctk1may play a role in cell cycle regulation (Myerson, EMBO J., 11(8):2909-17, 1992).

PCTAIRE-3

PCTAIRE-3 (Pctk3) is 65% homologous to Pctk1 and is expressed in brain,kidney and intestine (Okuda et al. Oncogene, 7(11):2249-58, 1992).

PFTAIRE Protein Kinase 1

PFTAIRE Protein Kinase 1 (Pftk1) is a cdk-related protein kinase andexhibits approximately 50% identity with Pctk3. Pftk1 is widelyexpressed in murine tissue and is though to play a role in meiosis andneuronal function (Besset et al., Mol. Reprod. Dev., 50(1): 18-29,1998).

Mitogen Activated Protein Kinase 9

Mitogen activated protein kinase 9 (Mapk9; also known as JNK2) caninhibit insulin signaling by stimulating phosphorylation of insulinreceptor substrate 1 (Irs 1) (Lee et al., J. Biol. Chem., 278(5):2896-902, 2003).

Positive Regulators of Glucose Transport

Increasing expression or activity of the genes listed in Table 2 canpotentiate insulin action by increasing insulin-stimulated glucoseuptake. As discussed for the negative regulators, above, potentiation ofinsulin action is beneficial, e.g., in controlling blood glucose actionin vivo. Thus, increasing the expression or activity of these geneproducts can be beneficial in the treatment of conditions in whichinsulin activity or glucose transport is disregulated such as diabetes.TABLE 2 Positive Regulators of Glucose Transport Gene or Gene ProductName Gene or Gene GenBank ® (abbreviated) Product Name Accession No.*Comments 9130022E05Rik AK078461 2900045N06Rik NM_028385 4930402E16RikAK129472 5730403B10Rik NM_025670 Abcfl ATP-binding BC063094 cassette,sub-family F D11Ertd498 BC024811 D19Ertd703e AK018652 F830029L24RikBC059190 G430055L02Rik BC015285 Crim1 Cysteine-rich motor XM_128751neuron 1 Dvl1 Dishevelled U10115 segment polarity protein homolog Lpin1Lipin AF180471 Nsdh1 NAD(P) dependent NM_010941 steroiddehydrogenase-like Ilk Integrin linked NM_010562 Kinase kinase Pard6gPar-6 partitioning NM_053117 defective 6 homolog gamma*GenBank ® entries in this table refer to murine sequences unlessotherwise noted.

9130022E05Rik

The gene product encoded by 9130022E05Rik, the nucleotide sequence ofwhich can be found under GenBank® Acc. No. AK078461, exhibits homologyto human regulatory solute carrier protein, family 1 (RSC1A1) andincludes a putative ubiquitin associated (UBA) domain.

2900045N06Rik

2900045N06Rik (found under GenBank® Acc. No. NM_(—)028385) encodes aputative SET methyl transferase domain and a putative ZnF_NFXtranscriptional repressor domain.

4930402E16Rik

4930402E16Rik (found under GenBank® Acc. No. AK129472) encodes domainswith homology to dimethylglycine dehydrogenase and glycine cleavageT-protein (GCV_T aminomethyl transferase) domains.

5730403B10Rik

The gene product encoded by 5730403B10Rik (found under GenBank® Acc. No.NM_(—)025670) encodes a putative LPS-induced tumor necrosis factor alphafactor (LITAF) membrane associated motif, also known as PIG7.

ATP-Binding Cassette, Sub-Family F, Member 1

The ATP-binding Cassette, Sub-family F (GCN20), Member 1 (Abcf1) genesequence is homologous to the human ABC50 sequence. Human ABC50 is anABC family member that appears to lack the transmembrane domains typicalof ABC transporters and may encode protein involved in translation ofmRNA (Richard et al., Genomics, 53(2):137-45, 1998).

Cysteine-Rich Motor Neuron 1

The Cysteine-rich Motor Neuron 1 gene (CRIM 1) encodes a transmembraneprotein containing an insulin-like growth factor (IGF)-binding proteinmotif and multiple cysteine-rich repeats (Kolle et al., Mech. Dev.,90(2): 181-193, 2000).

Dishevelled Segment Polarity Protein Homolog

The Dishevelled Segment Polarity Protein Homolog gene product (Dvl-1)has been implicated in microtubule assembly (Krylova et al., J. CellBiol., 151(1):83-94, 2000) and may regulate neurite outgrowth inconjunction with Wnt proteins (Kishida et al., Mol Cell Biol.,24(10):4487-501, 2004).

Lipin

The Lipin gene product (Lpin1) is required for induction of adipogenicgene transcription in mice, and mutations in the gene causelipodystrophy in the fatty liver dystrophy (fld) mouse (Phan et al., J.Biol. Chem., 279(28):29558-64, 2004).

NAD(P)H Steroid Dehydrogenase-Like Gene

NAD(P)H Steroid Dehydrogenase-like Gene (NSDHL) encodes a steroldehydrogenase or decarboxylase involved in post-squalene cholesterolbiosynthesis. Mutations in the human gene are associated with the humanCHILD syndrome (congenital hemidysplasia with ichthyosiform nevus andlimb defects) (Caldas and Herman, Hum. Mol., Genet., 12(22):2981-2991,2003). NSDHL is localized in the endoplasmic reticulum and associateswith lipid droplets (Caldas and Herman, supra).

Integrin Linked Kinase

Integrin Linked Kinase (Ilk) is a component of focal adhesions and bindsto the cytoplasmic tail of integrins, modulating actin rearrangements atintegrin adhesion sites (Sakai et al., Genes Dev., 17(7): 926-920,2003).

Par-6 Partitioning Defective 6 Homolog Gamma

Par-6 Partitioning Defective 6 Homolog Gamma (Pard6g) are similar to theC. elegans PDZ-domain protein PAR-6. Par6 is implicated in the formationof tight junctions at epithelial cell-cell contacts (Joberty et al.,Nat. Cell. Biol., 2(8): 531-539, 2000).

Screening Assays

Provided herein are methods for identifying modulators, i.e., candidateagents or reagents, of expression or activity of a glucosetransport-related nucleic acid or polypeptide. Such candidate agents orreagents include polypeptides, oligonucleotides, peptidomimetics,carbohydrates, or small molecules such as small organic or inorganicmolecules (e.g., non-nucleic acid small organic chemical compounds) thatmodulate expression (protein or mRNA) or activity of one or more glucosetransport-related polypeptides or nucleic acids. In general, screeningassays involve assaying the effect of a test agent on expression oractivity of a glucose transport-related nucleic acid or polypeptide in atest sample (i.e., a sample containing the glucose transport-relatednucleic acid or polypeptide). Expression or activity in the presence ofthe test compound or agent is compared to expression or activity in acontrol sample (i.e., a sample containing a glucose transport-relatedpolypeptide that was incubated under the same conditions, but withoutthe test compound). A change in the expression or activity of theglucose transport-related nucleic acid or polypeptide in the test samplecompared to the control indicates that the test agent or compoundmodulates expression or activity of the glucose transport-relatednucleic acid or polypeptide and is a candidate agent.

In one embodiment, the invention provides assays for screening testagents that bind to or modulate the activity of a glucosetransport-related polypeptide or nucleic acid encoding the polypeptideor biologically active portion thereof. The test compounds to bescreened, can be obtained using any of the numerous approaches incombinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the“one-bead one-compound” library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer, or small molecule librariesof compounds (Lam, Anticancer Drug Des., 12:145, 1997).

Examples of methods for the synthesis of molecular libraries can befound in the literature, for example in: DeWitt et al., Proc. Natl.Acad. Sci. USA, 90:6909, 1993; Erb et al., Proc. Natl. Acad. Sci. USA,91:11422, 1994; Zuckermann et al., J. Med. Chem. 37:2678, 1994; Cho etal., Science 261:1303, 1993; Carrell et al., Angew. Chem. Int. Ed. Engl.33:2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl., 33:2061,1994; and Gallop et al., J Med. Chem., 37:1233, 1994.

Libraries of compounds may be presented in solution (e.g., Houghten,Bio/Techniques, 13:412-421,1992), or on beads (Lam, Nature, 354:82-84,1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (U.S. Pat. No.5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409),plasmids (Cull et al., Proc. Natl. Acad. Sci. USA, 89:1865-1869, 1992)or phage (Scott and Smith, Science, 249:386-390, 1990; Devlin, Science,249:404-406, 1990; Cwirla et al., Proc. Natl. Acad. Sci. USA,87:6378-6382, 1990; and Felici, J. Mol. Biol., 222:301-310, 1991).

In one embodiment, the assay is a cell-based assay in which a cellexpressing a glucose transport-related polypeptide (e.g., a gene productof a gene Table 1 or 2), or a biologically active portion thereof, onthe cell surface is contacted with a test compound. The ability of thetest compound to bind to the polypeptide is then determined. The cell,for example, can be a yeast cell or a cell of mammalian origin. Theability of the test compound to bind to the polypeptide can bedetermined, for example, by coupling the test compound with aradioisotope or enzymatic label such that binding of the test compoundto the polypeptide or biologically active portion thereof can bedetermined by detecting the labeled compound in a complex. For example,test compounds can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, eitherdirectly or indirectly, and the radioisotope detected by direct countingof radioemmission or by scintillation counting.

Alternatively, test compounds can be enzymatically labeled with, forexample, horseradish peroxidase, alkaline phosphatase, or luciferase,and the enzymatic label detected by determination of conversion of anappropriate substrate to product. In one embodiment, the assay includescontacting a cell that expresses a membrane-bound form of the glucosetransport-related polypeptide, or a biologically active portion thereof,on the cell surface with a known compound that binds to the polypeptideto form an assay mixture, contacting the assay mixture with a testcompound, and determining the ability of the test compound to interactwith the polypeptide, e.g., by observing whether the test compoundpreferentially binds to the polypeptide or a biologically active portionthereof as compared to the known compound.

In another embodiment, an assay is a cell-based assay that includescontacting a cell expressing a membrane-bound form of a glucosetransport-related polypeptide, or a biologically active portion thereof,on the cell surface with a test compound and determining the ability ofthe test compound to modulate (e.g., stimulate or inhibit) the activityof the polypeptide or biologically active portion thereof. The abilityof the test compound to modulate the activity of the polypeptide or abiologically active portion thereof can be determined, for example, bymonitoring the ability of the polypeptide to bind to or interact with atarget molecule.

The ability of a polypeptide or nucleic acid to bind to or interact witha target molecule can be determined by one of the direct binding methodsdescribed herein. As used herein, a “target molecule” is a molecule withwhich a selected polypeptide or nucleic acid (e.g., a gene orpolypeptide encoded by a gene of Table 1 or Table 2, or a homologthereof) binds or interacts with in nature, for example, a molecule onthe surface of a cell that expresses the selected protein, a molecule onthe surface of a second cell, a molecule in the extracellular milieu, amolecule associated with the internal surface of a cell membrane, or acytoplasmic molecule. A target molecule can be a glucose transportrelated polypeptide or nucleic acid or some other polypeptide, protein,or nucleic acid. For example, a target molecule can be a component of asignal transduction pathway that facilitates transduction of anextracellular signal (e.g., a signal generated by binding of a compoundto a glucose transport-related polypeptide) through the cell membraneand into the cell or a second intercellular protein that has catalyticactivity, or a protein that facilitates the association of downstreamsignaling molecules with a glucose transport-related polypeptide.

The ability of a polypeptide to bind to or interact with a targetmolecule can also be determined. For example, the activity of the targetmolecule can be determined by detecting induction of a cellular secondmessenger of the target (e.g., intracellular Ca²⁺, diacylglycerol, orIP3), detecting catalytic/enzymatic activity of the target on anappropriate substrate (e.g., detecting kinase activity where the glucosetransport-related polypeptide is a kinase), detecting the induction of areporter gene (e.g., a regulatory element that is responsive to aglucose transport-related polypeptide operably linked to a nucleic acidencoding a detectable marker, e.g., luciferase), or detecting a cellularresponse, for example, cellular differentiation, or cell proliferation.When the target molecule is a nucleic acid, the compound can be, e.g., aribozyme or antisense molecule.

In yet another embodiment, an assay as described herein includescontacting a glucose transport-related polypeptide (e.g., a gene productof a gene of Table 1 or 2) or nucleic acid encoding the polypeptide, orbiologically active portion thereof, with a test compound anddetermining the ability of the test compound to bind to the polypeptideor biologically active portion thereof. Binding of the test compound tothe polypeptide can be determined either directly or indirectly asdescribed above. In one embodiment, the assay includes contacting thepolypeptide or biologically active portion thereof with a known compoundthat specifically binds the polypeptide to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with the polypeptide (e.g., itsability to compete with binding of the known compound). One can evaluatethe ability of the test compound to interact with the polypeptide bydetermining whether the test compound can preferentially bind to thepolypeptide or biologically active portion thereof as compared to theknown compound. When the test compound is targeted to a nucleic acid,the binding of the test compound to the nucleic acid can be tested,e.g., by binding, by fragmentation of the nucleic acid (as when the testcompound is a ribozyme), or by inhibition of transcription ortranslation in the presence of the test compound.

In another embodiment, an assay is a cell-free assay that includescontacting a glucose transport-related polypeptide biologically activeportion thereof with a test compound and determining the ability of thetest compound to modulate (e.g., stimulate or inhibit) the activity ofthe polypeptide or biologically active portion thereof. For example,determining the ability of the test compound to modulate the activity ofthe polypeptide can be accomplished by determining the ability of thepolypeptide to modify a target molecule. Such methods can,alternatively, measure the catalytic/enzymatic activity of the targetmolecule on an appropriate substrate. A number of the genes in Table 1and 2 encode kinases. For the products of these genes, one can utilizekinase assays to identify an agent that modulates the activity of thegene product. In general, modulation of an activity of the polypeptide(or a biologically portion thereof), by a kinase assay or another typeof assay, is determined by comparing the activity in the absence of thetest compound to the activity in the presence of the test compound. Ingeneral, modulation of an activity of the polypeptide (or a biologicallyportion thereof), by a kinase assay or another type of assay, isdetermined by comparing the activity in the absence of the test compoundto the activity in the presence of the test compound. For example, todetermine the activity of a kinase (e.g., a kinase listed in Table 1 orTable 2) in the presence of a test compound, any standard assay forprotein phosphorylation can be carried out. One can use a naturalsubstrate of the kinase or another protein or peptide that the kinasephosphorylates. Assays for kinase activity can also be carried out withbiologically active fragments of the kinase (e.g., a fragment thatretains catalytic activity).

In yet another embodiment, the cell-free assay includes contacting aglucose transport-related polypeptide or nucleic acid encoding thepolypeptide, or biologically active portion thereof, with a knowncompound that binds to the polypeptide to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with the polypeptide or nucleicacid by assaying the ability of the polypeptide or nucleic acid topreferentially bind to or modulate the activity of a target molecule(e.g., a target molecule that is a natural substrate or binding partnerof the polypeptide).

Cell-free assays are amenable to use of either a soluble form or amembrane-bound form of a polypeptide (if the polypeptide is amembrane-containing polypeptide. In the case of cell-free assayscomprising a membrane-bound form of the polypeptide, it may be desirableto utilize a solubilizing agent such that the membrane-bound form of thepolypeptide is maintained in solution. Examples of such solubilizingagents include non-ionic detergents such as n-octylglucoside,n-dodecylglucoside, n-octylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton X-100®, Triton X-114®, Thesit,Isotridecypoly(ethylene glycol ether)n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1 -propane sulfonate(CHAPSO), and N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate.

In certain embodiments of the new assay methods, it may be desirable toimmobilize either the glucose transport-related polypeptide or itstarget molecule to facilitate separation of complexed from uncomplexedforms of one or both of the proteins, as well as to automate the assay.Binding of a test compound to the polypeptide, or interaction of thepolypeptide with a target molecule in the presence and absence of a testagent, can be accomplished in any vessel suitable for containing thereactants. Examples of such vessels include microtiter plates, testtubes, and micro-centrifuge tubes. In one embodiment, a fusion proteincan be provided which adds a domain that allows one or both of theproteins to be bound to a matrix. For example, glutathione-S-transferasefusion proteins or glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the test compound or the test compound and either thenon-adsorbed target protein or a glucose-transport-related polypeptide,and the mixture incubated under conditions conducive to complexformation (e.g., at physiological conditions for salt and pH). Followingincubation, the beads or microtiter plate wells are washed to remove anyunbound components and complex formation is measured either directly orindirectly, for example, as described above. Alternatively, thecomplexes can be dissociated from the matrix, and the level of bindingor activity of the polypeptide can be determined using standardtechniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays. For example, either the glucosetransport-related polypeptide or its target molecule can be immobilizedutilizing conjugation of biotin and streptavidin. Biotinylatedpolypeptides or target molecules can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g.,biotinylation kit, Pierce Chemicals; Rockford, Ill.), and immobilized inthe wells of streptavidin-coated 96 well plates (Pierce Chemical).Alternatively, antibodies reactive with the polypeptide or targetmolecules but which do not interfere with binding of the glucosetransport-related polypeptide to its target molecule can be derivatizedto the wells of the plate, and unbound target or polypeptide trapped inthe wells by antibody conjugation. Methods for detecting such complexessuch as GST-immobilized complexes, include immunodetection of complexesusing antibodies reactive with the polypeptide or target molecule, aswell as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the polypeptide or target molecule.

In another embodiment, modulators of expression of a polypeptide areidentified in a method in which a cell is contacted with a test agent orcompound and the expression of the selected mRNA or protein (e.g., themRNA or protein corresponding to a glucose transport-related polypeptideor gene encoding the polypeptide, e.g., in Table 1 or 2) in the cell isdetermined. The level of expression of the selected mRNA or protein inthe presence of the test agent is compared to the level of expression ofthe selected mRNA or protein in the absence of the test agent. The testagent can then be identified as a modulator of expression of thepolypeptide (i.e., a candidate compound) based on this comparison. Forexample, when expression of the selected mRNA or protein is greater(statistically significantly greater) in the presence of the test agentthan in its absence, the test agent is identified as a candidate agentthat is a stimulator of the selected mRNA or protein expression.Alternatively, when expression of the selected mRNA or protein is less(statistically significantly less) in the presence of the test agentthan in its absence, the test agent is identified as a candidate agentthat is an inhibitor of the selected mRNA or protein expression. Thelevel of the selected mRNA or protein expression in the cells can bedetermined by methods described herein.

In yet another aspect, a glucose transport-related polypeptide can beused as a “bait protein” in a two-hybrid assay or three hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., Cell, 72:223-232,1993; Madura et al., J. Biol. Chem. 268:12046-12054, 1993; Bartel etal., Bio/Techniques 14:920-924, 1993; Iwabuchi et al., Oncogene8:1693-1696, 1993; and PCT Publication No. WO 94/10300), to identifyother proteins that bind to or interact with the glucosetransport-related polypeptide and modulate activity of the polypeptide.Such binding proteins are also likely to be involved in the propagationof signals by the glucose transport-related polypeptide as, for example,downstream elements of the signaling pathway involving glucosetransport.

Isolating Homologous Sequences from Other Species

The human homologs of glucose-transport related genes and their productsare useful for various embodiments of the present invention includingfor screening modulators and diagnosing of glucose transport-relateddisorders such as type II diabetes. Homologs have already beenidentified for certain genes. In those cases where a human homolog isnot identified, several approaches can be used to identify such genes.These methods include low stringency hybridization screens of humanlibraries with a mouse glucose transport-related nucleic acid sequence,polymerase chain reactions (PCR) of human DNA sequence primed withdegenerate oligonucleotides derived from a mouse glucosetransport-related gene, two-hybrid screens, and database screens forhomologous sequences.

Antisense Nucleic Acids

Agents to modulate the expression of the glucose transport-relatedpolypeptides described herein include antisense nucleic acid molecules,i.e., nucleic acid molecules whose nucleotide sequence is complementaryto all or part of an mRNA based on the sequence of a gene encodingglucose transport-related polypeptide (e.g., based on a sequence of agene of Tables 1 or 2). An antisense nucleic acid molecule can beantisense to all or part of a non-coding region of the coding strand ofa nucleotide sequence encoding the glucose transport-relatedpolypeptide. Non-coding regions (“5′ and 3′ untranslated regions”) arethe 5′ and 3′ sequences that flank the coding region in a gene and arenot translated into amino acids.

Based upon the sequences disclosed herein, one of skill in the art caneasily choose and synthesize any of a number of appropriate antisensemolecules to target a gene described herein. For example, a “gene walk”comprising a series of oligonucleotides of 15-30 nucleotides spanningthe length of a nucleic acid described in Table 1 can be prepared,followed by testing for inhibition of expression of the gene.Optionally, gaps of 5-10 nucleotides can be left between theoligonucleotides to reduce the number of oligonucleotides synthesizedand tested.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20,25, 30, 35, 40, 45, or 50 nucleotides or more in length. An antisensenucleic acid can be constructed using chemical synthesis and enzymaticligation reactions using procedures known in the art. For example, anantisense nucleic acid (e.g., an antisense oligonucleotide) can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused.

Examples of modified nucleotides which can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid(v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid(v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules described herein can be prepared invitro and administered to an animal, e.g., a mammal, e.g., a humanpatient. Alternatively, they can be generated in situ such that theyhybridize with or bind to cellular mRNA and/or genomic DNA encoding aselected polypeptide of the invention to thereby inhibit expression,e.g., by inhibiting transcription and/or translation. The hybridizationcan be by conventional nucleotide complementarities to form a stableduplex, or, for example, in the case of an antisense nucleic acidmolecule that binds to DNA duplexes, through specific interactions inthe major groove of the double helix. An example of a route ofadministration of antisense nucleic acid molecules includes directinjection at a tissue site. Alternatively, antisense nucleic acidmolecules can be modified to target selected cells and then administeredsystemically. For example, for systemic administration, antisensemolecules can be modified such that they specifically bind to receptorsor antigens expressed on a selected cell surface, e.g., by linking theantisense nucleic acid molecules to peptides or antibodies that bind tocell surface receptors or antigens. The antisense nucleic acid moleculescan also be delivered to cells using the vectors described herein. Forexample, to achieve sufficient intracellular concentrations of theantisense molecules, vector constructs can be used in which theantisense nucleic acid molecule is placed under the control of a strongpol II or pol III promoter.

An antisense nucleic acid molecule of the invention can be an α-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual, β-units, the strands run parallel to each other(Gaultier et al., Nucleic Acids Res. 15:6625-6641, 1987). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al., Nucleic Acids Res., 15:6131-6148, 1987) or a chimericRNA-DNA analog (Inoue et al., FEBS Lett., 215:327-330, 1987).

Antisense molecules that are complementary to all or part of a glucosetransport-related gene are also useful for assaying expression of suchgenes using hybridization methods known in the art. For example, theantisense molecule is labeled (e.g., with a radioactive molecule) and anexcess amount of the labeled antisense molecule is hybridized to an RNAsample. Unhybridized labeled antisense molecule is removed (e.g., bywashing) and the amount of hybridized antisense molecule measured. Theamount of hybridized molecule is measured and used to calculate theamount of expression of the glucose transport-related gene. In general,antisense molecules used for this purpose can hybridize to a sequencefrom a glucose transport-related gene under high stringency conditionssuch as those described herein. When the RNA sample is first used tosynthesize cDNA, a sense molecule can be used. It is also possible touse a double-stranded molecule in such assays as long as thedouble-stranded molecule is adequately denatured prior to hybridization.

Ribozymes

Also provided are ribozymes that have specificity for sequences encodingthe glucose transport-related polypeptides described herein (e.g., forsequences of the genes in Table 1 or 2). Ribozymes are catalytic RNAmolecules with ribonuclease activity that are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff and Gerlach, Nature, 334:585-591, 1988)) can beused to catalytically cleave mRNA transcripts to thereby inhibittranslation of the protein encoded by the mRNA. A ribozyme havingspecificity for a nucleic acid molecule of the invention can be designedbased upon the nucleotide sequence of a cDNA disclosed herein. Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved in a glucose transport-relatedmRNA (Cech et al. U.S. Pat. No. 4,987,071; and Cech et al., U.S. Pat.No. 5,116,742). Alternatively, an mRNA encoding a polypeptide of theinvention can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See, e.g., Barteland Szostak, Science, 261:1411-1418, 1993.

Also provided herein are nucleic acid molecules that form triple helicalstructures. For example, expression of a glucose transport-relatedpolypeptide can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the gene encoding thepolypeptide (e.g., the promoter and/or enhancer) to form triple helicalstructures that prevent transcription of the gene in target cells. Seegenerally Helene, Anticancer Drug Des. 6(6):569-84, 1991; Helene, Ann.N.Y. Acad. Sci., 660:27-36, 1992; and Maher, Bioassays, 14(12):807-15,1992.

In various embodiments, nucleic acid molecules (e.g., nucleic acidmolecules used to modulate expression of a glucose transport-relatedpolypeptide) can be modified at the base moiety, sugar moiety orphosphate backbone to improve, e.g., the stability, hybridization, orsolubility of the molecule. For example, the deoxyribose phosphatebackbone of the nucleic acids can be modified to generate peptidenucleic acids (see Hyrup et al., Bioorganic & Medicinal Chem., 4(1):5-23, 1996). Peptide nucleic acids (PNAs) are nucleic acid mimics, e.g.,DNA mimics, in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs allows for specific hybridizationto DNA and RNA under conditions of low ionic strength. The synthesis ofPNA oligomers can be performed using standard solid phase peptidesynthesis protocols, e.g., as described in Hyrup et al., 1996, supra;Perry-O'Keefe et al., Proc. Natl. Acad. Sci. USA, 93: 14670-675, 1996.

PNAs can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs canalso be used, e.g., in the analysis of single base pair mutations in agene by, e.g., PNA directed PCR clamping; as artificial restrictionenzymes when used in combination with other enzymes, e.g., S1 nucleases(Hyrup, 1996, supra; or as probes or primers for DNA sequence andhybridization (Hyrup, 1996, supra; Perry-O'Keefe et al., Proc. Natl.Acad. Sci. USA, 93: 14670-675, 1996).

PNAs can be modified, e.g., to enhance their stability or cellularuptake, by attaching lipophilic or other helper groups to PNA, by theformation of PNA-DNA chimeras, or by the use of liposomes or othertechniques of drug delivery known in the art. For example, PNA-DNAchimeras can be generated which may combine the advantageous propertiesof PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNAseH and DNA polymerases, to interact with the DNA portion while the PNAportion would provide high binding affinity and specificity. PNA-DNAchimeras can be linked using linkers of appropriate lengths selected interms of base stacking, number of bonds between the nucleobases, andorientation (Hyrup, 1996, supra). The synthesis of PNA-DNA chimeras canbe performed as described in Hyrup,1996, supra, and Finn et al., NucleicAcids Res., 24:3357-63, 1996. For example, a DNA chain can besynthesized on a solid support using standard phosphoramidite couplingchemistry and modified nucleoside analogs. Compounds such as5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite can be usedas a link between the PNA and the 5′ end of DNA (Mag et al., NucleicAcids Res., 17:5973-88, 1989). PNA monomers are then coupled in astepwise manner to produce a chimeric molecule with a 5′ PNA segment anda 3′ DNA segment (Finn et al., Nucleic Acids Res., 24:3357-63, 1996).Alternatively, chimeric molecules can be synthesized with a 5′ DNAsegment and a 3′ PNA segment (Peterser et al., Bioorganic Med. Chem.Lett., 5:1119-11124, 1975).

In some embodiments, the oligonucleotide includes other appended groupssuch as peptides (e.g., for targeting host cell receptors in vivo), oragents facilitating transport across the cell membrane (see, e.g.,Letsinger et al., Proc. Natl. Acad. Sci. USA, 86:6553-6556, 1989;Lemaitre et al., Proc. Natl. Acad. Sci. USA, 84:648-652, 1989; WO88/09810) or the blood-brain barrier (see, e.g., WO 89/10134). Inaddition, oligonucleotides can be modified with hybridization-triggeredcleavage agents (see, e.g., Krol et al., Bio/Techniques, 6:958-976,1988) or intercalating agents (see, e.g., Zon, Pharm. Res., 5:539-549,1988). To this end, the oligonucleotide may be conjugated to anothermolecule, e.g., a peptide, hybridization triggered cross-linking agent,transport agent, hybridization-triggered cleavage agent, etc.

siRNA

Another means by which expression of glucose transport-relatedpolypeptides can be inhibited is by RNA interference (RNAi). RNAi is aprocess in which mRNA is degraded in host cells. To inhibit an mRNA,double-stranded RNA (dsRNA) corresponding to a portion of the gene to besilenced (e.g., a gene encoding a glucose transport-related polypeptide,e.g., a gene of Table 1) is introduced into a cell. The dsRNA isdigested into 21-23 nucleotide-long duplexes called short interferingRNAs (or siRNAs), which bind to a nuclease complex to form what is knownas the RNA-induced silencing complex (or RISC). The RISC targets thehomologous transcript by base pairing interactions between one of thesiRNA strands and the endogenous mRNA. It then cleaves the mRNA about 12nucleotides from the 3′ terminus of the siRNA (see Sharp et al., GenesDev. 15:485-490, 2001, and Hammond et al., Nature Rev. Gen., 2:110-119,2001).

RNA-mediated gene silencing can be induced in mammalian cells in manyways, e.g., by enforcing endogenous expression of RNA hairpins (seePaddison et al., Proc. Natl. Acad. Sci. USA, 99:1443-1448, 2002) or, asnoted above, by transfection of small (21-23 nt) dsRNA (reviewed inCaplen, Trends in Biotech., 20:49-51, 2002). Methods for modulating geneexpression with RNAi are described, e.g., in U.S. Pat. No. 6,506,559 andU.S. Pat. Pub. No. 20030056235, which are hereby incorporated byreference.

Standard molecular biology techniques can be used to generate siRNAs.Short interfering RNAs can be chemically synthesized, recombinantlyproduced, e.g., by expressing RNA from a template DNA, such as aplasmid, or obtained from commercial vendors such as Dharmacon. The RNAused to mediate RNAi can include synthetic or modified nucleotides, suchas phosphorothioate nucleotides. Methods of transfecting cells withsiRNA or with plasmids engineered to make siRNA are routine in the art.

The siRNA molecules used to modulate expression of a glucosetransport-related polypeptide can vary in a number of ways. For example,they can include a 3′ hydroxyl group and strands of 21, 22, or 23consecutive nucleotides. They can be blunt ended or include anoverhanging end at either the 3′ end, the 5′ end, or both ends. Forexample, at least one strand of the RNA molecule can have a 3′ overhangfrom about 1 to about 6 nucleotides (e.g., 1-5, 1-3, 2-4 or 3-5nucleotides (whether pyrimidine or purine nucleotides) in length. Whereboth strands include an overhang, the length of the overhangs may be thesame or different for each strand.

To further enhance the stability of the RNA duplexes, the 3′ overhangscan be stabilized against degradation (by, e.g., including purinenucleotides, such as adenosine or guanosine nucleotides or replacingpyrimidine nucleotides by modified analogues (e.g., substitution ofuridine 2 nucleotide 3′ overhangs by 2′-deoxythymidine is tolerated anddoes not affect the efficiency of RNAi). Any siRNA can be used in themethods of modulating a glucose transport-related polypeptide, providedit has sufficient homology to the target of interest. There is no upperlimit on the length of the siRNA that can be used (e.g., the siRNA canrange from about 21 base pairs of the gene to the full length of thegene or more (e.g., 50-100, 100-250, 250-500, 500-1000, or over 1000base pairs).

Isolated Polypeptides

Isolated polypeptides encoded by the glucose transport-related genesdescribed herein are also provided. These polypeptides can be used,e.g., as immunogens to raise antibodies, in screening methods, or inmethods of treating subject, e.g., by administration of thepolypeptides. Methods are well known in the art for predicting thetranslation products of the nucleic acids (e.g., using computer programsthat provide the predicted polypeptide sequences and direction as towhich of the three reading frames is the open reading frame of thesequence). These polypeptide sequences can then be produced eitherbiologically (e.g., by positioning the nucleic acid sequence thatencodes them in-frame in an expression vector transfected into acompatible expression system) or chemically using methods known in theart. The entire polypeptide or a fragment thereof can be used in amethod of treatment or to produce an antibody, e.g., that is useful in ascreening assay.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theprotein is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. Thus, protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofheterologous protein (also referred to herein as “contaminatingprotein”). In general, when the protein or biologically active portionthereof is recombinantly produced, it is also substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the protein preparation. In general, whenthe protein is produced by chemical synthesis, it is substantially freeof chemical precursors or other chemicals, i.e., it is separated fromchemical precursors or other chemicals that are involved in thesynthesis of the protein. Accordingly such preparations of the proteinhave less than about 30%, 20%, 10%, or 5% (by dry weight) of chemicalprecursors or compounds other than the polypeptide of interest.

Expression of proteins and polypeptides can be assayed to determine theamount of expression. Methods for assaying protein expression are knownin the art and include Western blot, immunoprecipitation, andradioimmunoassay.

Biologically active portions of a glucose transport-related polypeptideinclude polypeptides comprising amino acid sequences sufficientlyidentical to or derived from the amino acid sequence of the protein,which include fewer amino acids than the full length protein, andexhibit at least one activity of the corresponding full-length protein.Typically, biologically active portions comprise a domain or motif withat least one activity of the corresponding protein. A biologicallyactive portion of a polypeptide can be, for example, 10, 25, 50, 100, ormore amino acids in length. Moreover, biologically active portions, inwhich other regions of a given protein are deleted, can be prepared byrecombinant techniques and evaluated for one or more of the functionalactivities of the native form of a polypeptide. These short polypeptidescan be used in treatments to competitively inhibit activity of the geneproducts of the genes listed in Tables 1 and 2.

In some embodiments, glucose transport-related polypeptides have thepredicted amino acid sequence encoded by a gene selected from the genesin Tables 1 and 2. Other useful proteins are substantially identical(e.g., at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, or 99%)to the predicted amino acid sequence of a polypeptide encoded by a genein Tables 1 and 2, and (a) retain the functional activity of the proteinof the corresponding naturally-occurring protein yet differ in aminoacid sequence due to natural allelic variation or mutagenesis, or (b)exhibit an altered functional activity (e.g., as a dominant negative)where desired.

The comparison of sequences and determination of percent identitybetween two sequences is accomplished using a mathematical algorithm.The percent identity between two amino acid sequences is determinedusing the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 )algorithm, which has been incorporated into the GAP program in the GCGsoftware package (available at http://www.gcg.com), using either aBlossum 62 matrix or a PAM250 matrix, and a gap weight of 16 and alength weight of 1. The percent identity between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage (available at http://www.gcg.com), using a NWSgapdna.CMP matrixand a gap weight of 40 and a length weight of 1.

In general, percent identity between amino acid sequences referred toherein is determined using the BLAST 2.0 program, which is available tothe public at http://www.ncbi.nlm.nih.gov/BLAST. Sequence comparison isperformed using an ungapped alignment and using the default parameters(Blossum 62 matrix, gap existence cost of 11, per residue gap cost of 1,and a lambda ratio of 0.85). The mathematical algorithm used in BLASTprograms is described in Altschul et al., Nucleic Acids Research25:3389-3402, 1997.

Also provided herein are chimeric or fusion proteins. As used herein, a“chimeric protein” or “fusion protein” comprises all or part (e.g., abiologically active portion) of a glucose transport-related polypeptideoperably linked to a heterologous polypeptide (i.e., a polypeptide otherthan the glucose transport-related polypeptide). In the context of afusion protein, the term “operably linked” is intended to indicate thatthe polypeptide and the heterologous polypeptide are fused in-frame toeach other. The heterologous polypeptide can be fused to the N-terminusor C-terminus of the glucose transport-related polypeptide.

One useful fusion protein is a GST fusion protein in which the glucosetransport-related polypeptide is fused to the C-terminus of GSTsequences. Such fusion proteins can facilitate the purification of arecombinant glucose transport-related polypeptide.

In another embodiment, the fusion protein contains a heterologous signalsequence at its N-terminus. For example, the native signal sequence of aglucose transport-related polypeptide can be removed and replaced with asignal sequence from another protein. For example, the gp67 secretorysequence of the baculovirus envelope protein can be used as aheterologous signal sequence (Current Protocols in Molecular Biology,Ausubel et al., eds., John Wiley & Sons, 1992). Other examples ofeukaryotic heterologous signal sequences include the secretory sequencesof melittin and human placental alkaline phosphatase (Stratagene; LaJolla, Calif.). In yet another example, useful prokaryotic heterologoussignal sequences include the phoA secretory signal (Sambrook et al.,eds., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989) and the protein A secretory signal (PharmaciaBiotech; Piscataway, N.J.).

In yet another embodiment, the fusion protein is an immunoglobulinfusion protein in which all or part of a glucose transport-relatedpolypeptide is fused to sequences derived from a member of theimmunoglobulin protein family. The immunoglobulin fusion proteins can beincorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a ligand (soluble ormembrane-bound) and a protein on the surface of a cell (receptor), tothereby suppress signal transduction in vivo. The immunoglobulin fusionprotein can be used to affect the bioavailability of a cognate ligand ofa glucose transport-related polypeptide. Inhibition of ligand/receptorinteraction may be useful therapeutically, both for treatingproliferative and differentiative disorders and for modulating (e.g.,promoting or inhibiting) cell survival. Moreover, the immunoglobulinfusion proteins can be used as immunogens to produce antibodies directedagainst a glucose transport-related polypeptide in a subject, to purifyligands and in screening assays to identify molecules that inhibit theinteraction of receptors with ligands.

Chimeric and fusion glucose transport-related polypeptides can beproduced by standard recombinant DNA techniques. In another embodiment,the fusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed and reamplified to generate a chimeric genesequence. Moreover, many expression vectors are commercially availablethat already encode a fusion moiety (e.g., a GST polypeptide). A nucleicacid encoding a glucose transport-related polypeptide can be cloned intosuch an expression vector such that the fusion moiety is linked in-frameto the polypeptide.

A signal sequence of a polypeptide can be used to facilitate secretionand isolation of the secreted protein or other proteins of interest.Signal sequences are typically characterized by a core of hydrophobicamino acids which are generally cleaved from the mature protein duringsecretion in one or more cleavage events. Such signal peptides containprocessing sites that allow cleavage of the signal sequence from themature proteins as they pass through the secretory pathway. Thus, thedescribed polypeptides having a signal sequence, as well as to thesignal sequence itself and to the polypeptide in the absence of thesignal sequence (i.e., the cleavage products), are provided herein. Inone embodiment, a nucleic acid sequence encoding a signal sequence canbe operably linked in an expression vector to a protein of interest,such as a protein which is ordinarily not secreted or is otherwisedifficult to isolate. The signal sequence directs secretion of theprotein, such as from a eukaryotic host into which the expression vectoris transformed, and the signal sequence is subsequently or concurrentlycleaved. The protein can then be readily purified from the extracellularmedium by methods known in the art. Alternatively, the signal sequencecan be linked to the protein of interest using a sequence whichfacilitates purification, such as with a GST domain.

Also provided are variants of the glucose transport-relatedpolypeptides. Such variants have an altered amino acid sequence whichcan function as either agonists (mimetics) or as antagonists. Variantscan be generated by mutagenesis, e.g., discrete point mutation ortruncation. An agonist can retain substantially the same, or a subset,of the biological activities of the naturally occurring form of theprotein. An antagonist of a protein can inhibit one or more of theactivities of the naturally occurring form of the protein by, forexample, competitively binding to a downstream or upstream member of acellular signaling cascade which includes the protein of interest. Thus,specific biological effects can be elicited by treatment with a variantof limited function. Treatment of a subject with a variant having asubset of the biological activities of the naturally occurring form ofthe protein can have fewer side effects in a subject relative totreatment with the naturally occurring form of the protein.

Antibodies

An isolated glucose transport-related polypeptide, or a fragmentthereof, can be used as an immunogen to generate antibodies usingstandard techniques for polyclonal and monoclonal antibody preparation.The full-length polypeptide or protein can be used or, alternatively,antigenic peptide fragments can be used as immunogens. The antigenicpeptide of a protein comprises at least 8 (e.g., 10, 15, 20, or 30)amino acid residues of the amino acid sequence of a glucosetransport-related polypeptide, e.g., encoded by a gene in Table 1 orTable 2, and encompasses an epitope of the protein such that an antibodyraised against the peptide forms a specific immune complex with theprotein.

An immunogen typically is used to prepare antibodies by immunizing asuitable subject, (e.g., rabbit, goat, mouse or other mammal). Anappropriate immunogenic preparation can contain, for example, arecombinantly expressed or a chemically synthesized polypeptide. Thepreparation can further include an adjuvant, such as Freund's completeor incomplete adjuvant, or similar immunostimulatory agent.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a glucose transport-related polypeptide as animmunogen. The antibody titer in the immunized subject can be monitoredover time by standard techniques, such as with an enzyme linkedimmunosorbent assay (ELISA) using immobilized polypeptide. If desired,the antibody molecules can be isolated from the mammal (e.g., from theblood) and further purified by well-known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the specific antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein, Nature256:495-497, 1975, the human B cell hybridoma technique (Kozbor et al.,Immunol. Today 4:72, 1983), the EBV-hybridoma technique (Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96,1985) or trioma techniques. The technology for producing hybridomas iswell known (see generally Current Protocols in Immunology, 1994, Coliganet al. (eds.) John Wiley & Sons, Inc., New York, N.Y.). Hybridoma cellsproducing a monoclonal antibody are detected by screening the hybridomaculture supernatants for antibodies that bind the polypeptide ofinterest, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody directed against a polypeptide can be identified andisolated by screening a recombinant combinatorial immunoglobulin library(e.g., an antibody phage display library) with the polypeptide ofinterest. Kits for generating and screening phage display libraries arecommercially available (e.g., the Pharmacia Recombinant Phage AntibodySystem, Catalog No. 27-9400-01; and the Stratagene SurfZAP™ PhageDisplay Kit, Catalog No. 240612). Additionally, examples of methods andreagents particularly amenable for use in generating and screeningantibody display library can be found in, for example, U.S. Pat. No.5,223,409; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO93/01288; WO 92/01047; WO 92/09690; WO 90/02809; Fuchs et al.,Bio/Technology 9:1370-1372, 1991; Hay et al., Hum. Antibod. Hybridomas3:81-85, 1992; Huse et al., Science 246:1275-1281, 1989; Griffiths etal., EMBO J. 12:725-734, 1993.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, areprovided herein. Such chimeric and humanized monoclonal antibodies canbe produced by recombinant DNA techniques known in the art, for exampleusing methods described in WO 87/02671; European Patent Application184,187; European Patent Application 171,496; European PatentApplication 173,494; WO 86/01533; U.S. Pat. No. 4,816,567; EuropeanPatent Application 125,023; Better et al., Science, 240:1041-1043, 1988;Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443, 1987; Liu et al.,J. Immunol., 139:3521-3526, 1987; Sun et al., Proc. Natl. Acad. Sci.USA, 84:214-218, 1987; Nishimura et al., Canc. Res. 47:999-1005, 1987;Wood et al., Nature, 314:446-449, 1985; and Shaw et al., J. Natl. CancerInst., 80:1553-1559, 1988); Morrison, Science, 229:1202-1207, 1985; Oiet al., Bio/Techniques, 4:214, 1986; U.S. Pat. No. 5,225,539; Jones etal., Nature, 321:552-525, 1986; Verhoeyan et al., Science, 239:1534,1988; and Beidler et al., J. Immunol., 141:4053-4060, 1988.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice which are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of apolypeptide. Monoclonal antibodies directed against the antigen can beobtained using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (Int. Rev. Immunol., 13:65 -93, 1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806. Inaddition, companies such as Abgenix, Inc. (Freemont, Calif.), can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Biotechnology,12:899-903, 1994).

An antibody directed against a glucose transport-related polypeptide(e.g., monoclonal antibody) can be used to isolate the polypeptide bystandard techniques, such as affinity chromatography orimmunoprecipitation. Moreover, such an antibody can be used to detectthe protein (e.g., in a cellular lysate or cell supernatant) in order toevaluate the abundance and pattern of expression of the polypeptide. Theantibodies can also be used diagnostically to monitor protein levels intissue as part of a clinical testing procedure, e.g., for example,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidinibiotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Methods of Treatment and Pharmaceutical Compositions

Methods of treating disorders related to glucose metabolism are providedherein. “Treating” includes methods that cure, alleviate, relieve,alter, ameliorate, palliate, improve or affect the disorder, thesymptoms of the disorder or the predisposition toward the disorder. Themethods can be used in vivo or on cells in culture, e.g., in vitro or exvivo. For in vivo embodiments, the method is effected in a subject andincludes administering the agent to the subject under conditionseffective to permit the agent to modulate the expression or activity ofthe polypeptide in a cell.

Agents that modulate expression or activity of a glucosetransport-related polypeptide in vitro are further tested in vivo inanimal models. For example, a test compound identified as a modulator ofa glucose transport-related polypeptide is tested in an animal such asan animal model for obesity or diabetes (e.g., type II diabetes, e.g.,ob/ob mice obtained from Jackson Laboratories (Strain Name:B6.V-Lep^(ob)/J), db/db mice; see, e.g., Sima A A F, Shafrir E. AnimalModels in Diabetes: A Primer. Taylor and Francis, Publ Amsterdam,Netherlands, 2000). At various time points after administration of thetest agent, levels of expression or activity of the glucosetransport-related polypeptide and/or levels of glucose, glucosetolerance, and plasma insulin are monitored to determine whether thetest compound has a beneficial effect on glucose metabolism, relative tocontrol, i.e., whether the test compound causes a reduction inhyperglycemia or plasma insulin levels.

Data obtained from the cell culture assays and animal studies can beused in formulating an appropriate dosage of any given agent for use inhumans. A therapeutically effective amount of an agent will be an amountthat delays progression of or improves one or more symptoms of thecondition, whether evident by improvement in an objective sign (e.g.,blood glucose levels) or subjective symptom of the disease. Certainfactors may influence the dosage and timing required to effectivelytreat a subject (e.g., the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present).

Compositions useful for modulating expression or activity of the glucosetransport-related polypeptides (whether previously known or identifiedby the screening assays described herein), can be incorporated intopharmaceutical compositions and administered to subjects who have, orwho are at risk of developing, a disorder or condition related toglucose metabolism (e.g., related to disregulated glucose metabolismsuch as type I diabetes, type II diabetes, or obesity). Suchcompositions will include one or more agents that modulate theexpression or activity of the glucose transport-related polypeptide anda pharmaceutically acceptable carrier (e.g., a solvent, dispersionmedium, coating, buffer, absorption delaying agent, and the like, thatare substantially non-toxic). Supplementary active compounds can also beincorporated into the compositions.

Pharmaceutical compositions are formulated to be compatible with theirintended route of administration, whether oral or parenteral (e.g.,intravenous, intradermal, subcutaneous, transmucosal (e.g., nasal spraysare formulated for inhalation), or transdermal (e.g., topical ointments,salves, gels, patches or creams as generally known in the art). Thecompositions can include a sterile diluent (e.g., sterile water orsaline), a fixed oil, polyethylene glycol, glycerine, propylene glycolor other synthetic solvents; antibacterial or antifungal agents such asbenzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates; and isotonic agentssuch as sugars (e.g., dextrose), polyalcohols (e.g., manitol orsorbitol), or salts (e.g., sodium chloride). Liposomal suspensions(including liposomes targeted to affected cells with monoclonalantibodies specific for neuronal antigens) can also be used aspharmaceutically acceptable carriers (see, e.g., U.S. Pat. No.4,522,811). Preparations of the compositions can be formulated andenclosed in ampules, disposable syringes or multiple dose vials. Whererequired (as in, for example, injectable formulations), proper fluiditycan be maintained by, for example, the use of a coating such aslecithin, or a surfactant. Absorption of the active ingredient can beprolonged by including an agent that delays absorption (e.g., aluminummonostearate and gelatin). Alternatively, controlled release can beachieved by implants and microencapsulated delivery systems, which caninclude biodegradable, biocompatible polymers (e.g., ethylene vinylacetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters,and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

Where oral administration is intended, the agent can be included inpills, capsules, troches and the like and can contain any of thefollowing ingredients, or compounds of a similar nature: a binder suchas microcrystalline cellulose, gum tragacanth or gelatin; an excipientsuch as starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate; aglidant such as colloidal silicon dioxide; a sweetening agent such assucrose or saccharin; or a flavoring agent such as peppermint, methylsalicylate, or orange flavoring.

Compositions containing the agents that modulate glucosetransport-related polypeptides can be formulated for oral or parenteraladministration in dosage unit form (i.e., physically discrete unitscontaining a predetermined quantity of active compound for ease ofadministration and uniformity of dosage). Toxicity and therapeuticefficacy of compounds can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals. One can, forexample, determine the LD₅₀ (the dose lethal to 50% of the population)and the ED₅₀ (the dose therapeutically effective in 50% of thepopulation), the therapeutic index being the ratio of LD₅₀:ED₅₀. Agentsthat exhibit high therapeutic indices are preferred. Where an agentexhibits an undesirable side effect, care should be taken to target thatagent to the site of the affected tissue (the aim being to minimizepotential damage to unaffected cells and, thereby, reduce side effects).Toxicity and therapeutic efficacy can be determined by other standardpharmaceutical procedures.

Data obtained from the cell culture assays and animal studies can beused in formulating an appropriate dosage of any given agent for use inhumans. A therapeutically effective amount of an agent will be an amountthat delays progression of or improves one or more symptoms of thecondition, whether evident by improvement in an objective sign (e.g.,blood glucose levels) or subjective symptom of the disease. Certainfactors may influence the dosage and timing required to effectivelytreat a subject (e.g., the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present).

As noted above, agents identified and administered according to themethods described here can be small molecules (e.g., peptides,peptidomimetics (e.g., peptoids), amino acid residues (or analogsthereof), polynucleotides (or analogs thereof), nucleotides (or analogsthereof), or organic or inorganic compounds (e.g., heteroorganic ororganometallic compounds). Typically, such molecules will have amolecular weight less than about 10,000 grams per mole (e.g., less thanabout 7,500, 5,000, 2,500, 1,000, or 500 grams per mole). Salts, esters,and other pharmaceutically acceptable forms of any of these compoundscan be assayed and, if a desirable activity is detected, administeredaccording to the therapeutic methods described herein. Exemplary dosesinclude milligram or microgram amounts of the small molecule perkilogram of subject or sample weight (e.g., about 1 μg-500 mg/kg; about100 μg-500 mg/kg; about 100 μg-50 mg/kg; 10μg-5 mg/kg; 10 μg-0.5 mg/kg;or 1 μg-50 μg/kg). While these doses cover a broad range, one ofordinary skill in the art will understand that therapeutic agents,including small molecules, vary in their potency, and effective amountscan be determined by methods known in the art. Typically, relatively lowdoses are administered at first, and the attending physician orveterinarian (in the case of therapeutic application) or a researcher(when still working at the clinical development stage) can subsequentlyand gradually increase the dose until an appropriate response isobtained. In addition, it is understood that the specific dose level forany particular subject will depend upon a variety of factors includingthe activity of the specific compound employed, the age, body weight,general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the degree of expression or activity to bemodulated.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The invention will be further described in the following examples whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Identification of Genes and Gene Products thatEnhance Insulin Action on Glucose Transport Using RNAi-Screening

Cell Culture and Electroporation of 3T3-L1 Adipocytes with siRNAOligonucleotides.

3T3-L1 fibroblasts were grown in DMEM supplemented with 10% FBS, 50μg/ml streptomycin, and 50 units/ml penicillin and differentiated intoadipocytes as described (Jiang et al., Proc. Natl. Acad. Sci. USA,100:7569-7574, 2003; Guilherrne et al., J. Biol. Chem., 279:10593-10605,2004). The 3T3-L1 adipocytes were transfected with siRNA duplexes byelectroporation. Briefly, 4 or 5 days after differentiation wasinitiated, adipocytes were detached from culture dishes with 0.25%trypsin and 0.5 mg of collagenase/ml in PBS and washed twice with PBS.Cells were washed, counted and resuspended at a density of 9×10⁶cells/ml in PBS. Typically, one 150 mm dish was transfected with 6different siRNA-duplexes. Resuspended cells (0.15 ml) were placed in 0.2cm gap cuvette (Bio-Rad®) and mixed with 4 nmoles of each SMARTpool®siRNA-duplexes, purchased from Dharmacon. siRNA oligonucleotides weredelivered to the cells by a pulse of electroporation with a Bio-Rad®gene pulser II system at the setting of 0.09 kV and 950 μF capacitance.After electroporation, cells were immediately mixed with 1 ml of freshcomplete DMEM media. Cells were then transferred from the cuvette to 3ml of DMEM media in a 15 ml Falcon™ tube and mixed. Aliquots (125 μl) ofthis cell suspension were seeded into wells of a 96 well plate. Cellsfrom each electroporation were spread into 12 such wells, placed in anincubator and 2-deoxyglucose uptake was measured 72 hours later. Theremainder of the cell suspension was distributed equally into 2 wells ofa 12 well plate for performing western blot and/or evaluating MAPK andAkt phosphorylation.

For each 2-deoxyglucose assay, cells were also electroporated withscrambled (6 nmoles), Akt1 and Akt2 (4 and 6 nmoles) and PTEN (6 nmoles)siRNAs, as controls. Each 96 well plate contained 12 wells of each ofthese 3 controls.

2-Deoxyglucose Uptake Assays

Insulin-stimulated glucose transport in 3T3-L1 adipocytes was estimatedby measuring 2-deoxyglucose uptake as described (Guilherme et al., JBiol Chem, 279:10593-10605, 2004). Briefly, siRNA transfected cells werereseeded on 96-well plates and cultured for 72 hours, washed twice andserum-starved for two hours with Krebs-Ringer's Hepes buffer (130 mMNaCl, 5 mM KCl, 1.3 mM CaCl₂, 1.3 mM MgSO₄, 25 mM Hepes, pH 7.4)supplemented with 0.5% BSA and 2 mm sodium pyruvate. Cells were thenstimulated with insulin for 30 minutes at 37° C. Glucose uptake wasinitiated by addition of [1,2-³H] 2-deoxy-D-glucose to a final assayconcentration of 500 μM. Cells were incubated for 5 minutes at 37° C.Assays were terminated by three washes with ice-cold Krebs-Ringer'sHepes buffer. Briefly, the plates were dipped into a container with 600ml of ice-cold Krebs-Ringer's Hepes buffer. Liquids in the wells weredrained after each dip by patting the plate on a wad of paper towels.The cells were then solubilized by adding 0.05 ml of 1% SDS per well.Uptake of [³H] was quantitated using a Microplate scintillation countinginstrument. Specific uptake was measured by determining non-specificdeoxyglucose uptake in samples incubated in the presence of 20 μMcytochalasin B and subtracting the values obtained for this control fromeach experimental determination.

Small inhibitory RNAs for approximately 500 different genes wereobtained and transfected into adipocytes and tested in deoxyglucoseuptake assays described above. A number of genes, when knocked down bytransfection of siRNA, resulted in increased or decreased glucose uptakeas compared to controls. The results of experiments in which 58different genes were targeted are depicted in FIG. 1.

The 58 candidate protein kinases selected for gene silencing wereidentified using Affymetrix GeneChip® array analysis of mRNA isolatedfrom 3T3-L1 preadipocytes versus fully differentiated 3T3-L1 adipocytesusing the Bioconductor statistical program, specifically rma and mas5,an implementation of the Affymetrix GeneChip® analysis program(Gentleman et al., Genome Biol. 5:R80, 2004). Briefly, total RNA wasisolated from 3T3-L1 fibroblast cells grown in culture for 7 days to aquiescent state (Adipocyte Day 0 Differentiation) or from 3T3-L1adipocytes at day 6, post addition of differentiation media. RNA wasisolated from three different days for each replicate. cRNA wasfragmented and hybridized to Affymetrix GeneChip® Mouse Expression Set430 A and 430 B arrays. Raw expression data were analyzed with theBioconductor statistical environment (Gentleman et al., Genome Biol.5:R80, 2004) using rma (Bolstad et al., Bioinformatics, 19:185, 2003)and mas5, a Bioconductor implementation of the MAS 5.0 algorithm(Affymetrix, available on the internet at affymetrix.com). The mas5program was applied to calculate a present or absent call for eachprobeset on a GeneChip. The calculation of these calls are based on aWilcoxon rank test between the PM and MM probes of a probeset. Only thekinases which showed a present call in each of the replicatehybridizations were filtered and used in subsequent analyses.

All kinases were considered expressed in adipocytes if they had mas5presence calls in each of triplicate hybridizations. Pools of 4 siRNAsequences directed against protein kinases were electroporated into3T3-L1 cells, and deoxyglucose transport assays on the transfectedadipocytes were performed 72 hours later in 96 well plates. In theseexperiments, scrambled siRNA was used as a control, and siRNA directedagainst Akt2 protein kinase and the PtdIns(3,4,5)P3 phosphatase PTEN,which function as positive and negative regulators of insulin signaling,respectively, were included.

RNAs targeting the following kinases listed in FIG. 1 caused increasedglucose uptake: IKKα, IKKβ, Map4k4, PCTK1, and PFTK1 (marked in FIG. 1with arrows). Depletion of one kinase, ILK, resulted in decreased uptake(marked with an asterisk in FIG. 1).

To confirm that siRNA-based gene silencing enhanced insulin-stimulated2-deoxyglucose uptake in adipocytes, further experiments were performedin which the targets identified by screening were independentlyretested. The results for experiments with IKKβ, IKKα, ILK, Map4k4,PCTK1, and PFTK1 are depicted in FIG. 2, with scrambled siRNA, AKT2, andPTEN siRNAs used as controls. ILK siRNA inhibited glucose uptake and theother siRNAs increased glucose uptake at both submaximal and maximaldoses of insulin in 3T3-L1 adipocytes, confirming the results obtainedin previous assays.

In order to further confirm and validate functionality of these proteinkinases in cultured adipocytes, a secondary siRNA-based screen wasconducted to confirm silencing efficiency and biological effects.PCTAIRE-1, IKKβ and IKKα proteins were substantially decreased byrespective siRNA treatments, as determined by Western blot analysis.Similarly, levels of mRNA encoding MAP4K4/NIK and PFTAIRE-1 weredecreased by siRNA treatment of cells, as detected by RT-PCR or realtime PCR.

Real time PCR was performed as follows. Briefly, total RNA was extractedfrom the cultured 3T3-L1 adipocytes using Trizol® (Invitrogen). TheSuperScript® one-step RT-PCR kit (Invitrogen) was used for RT-PCR. Thelower number of cycles was selected to avoid the polymerase chainreaction (PCR) entering plateau stages. For quantitative mRNA analysis,1 μg of total RNA was reverse transcribed using Bio-Rad's iScript cDNASynthesis kit (Bio-Rad). Ten percent of each RT reaction was subjectedto quantitative real-time PCR analysis using Bio-Rad's iQ SYBR greensupermix kit and Real-Time PCR detection system following manufacturer'sinstructions (MyiQ, Bio-Rad). We designed specific primer pair yieldingshort PCR product using an online database, PrimerBank (available on theinternet at pga.mgh.harvard.edu/primerbank) (Wang and Seed, NucleicAcids Res., 31(24):el54, 2003). Hyperxanthine-GuantinePhosphoribosyltransferase (HPRT) was used as standard housekeeping gene.Relative gene expression was calculated by subtracting the thresholdcycle number (Ct) of the target genes from the Ct value of HPRT andraising 2 to the power of this difference.

Genes for which siRNA targeting resulted in strongly increased anddecreased glucose uptake are listed in Tables 1 and 2, above.

Example 2 Characterization of Genes and Gene Products that ModulateGlucose Transport

Western Blotting

3T3-L1 adipocytes electroporated with indicated siRNA were starvedovernight in serum-free DMEM media. Cells were then incubated without orwith the indicated insulin concentrations for 30 minutes and harvestedwith lysis buffer containing 1% SDS. Protein concentrations werequantitated and equivalent amounts of protein from each lysate samplewere resolved by SDS-PAGE and transferred to nitrocellulose membranes.Membranes were incubated with antibodies overnight at 4° C. and thenwith horseradish peroxidase-linked secondary antibodies for 45 minutesat room temperature. Proteins were then detected with an enhancedchemiluminescence kit. For experiments described herein, the anti-PTEN,anti-Akt2, anti-Akt, anti-pAkt and anti-lamin A/C antibodies wereobtained as described (Jiang et al., Proc. Natl. Acad. Sci. U S. A.,100:7569-7574, 2003). Goat anti-GLUT4, rabbit anti-C/EBPα, anti-C/EBPβ,anti-PCTAIRE-1, mouse anti-PPARγ and anti-SREBP-1 antibodies were fromSanta Cruz Biotechnology. Rabbit anti-IKKα and anti-IKKβ were from CellSignaling Technology (Beverly, Mass.). Mouse monoclonal anti TATA bidingprotein (TBP) was from Abacam Inc.

Akt Phosphorylation

The effects of a subset of siRNAs on insulin-induced Akt phosphorylationwere examined by Western blotting, as described above. It has been shownthat Akt mediates insulin signaling (see Jiang et al., Proc. Natl. Acad.Sci. USA, 100:7569-7574, 2003; and references cited therein).Phosphorylation of Akt at serine 473 is indicative of activation.Adipocytes transfected with scrambled, IKKα, IKKβ, PTEN, PCTK1, ILK,Map4k4, or PFTK1 siRNAs were starved and stimulated with insulin at 0,1, and 100 nM concentrations. Lysates were resolved by SDS-PAGE andphosphorylation of serine 473 of Akt was analyzed by Western blot. Thequantification of the blots is depicted in FIG. 3. IKKβ and PTEN siRNAsenhanced insulin-induced phosphorylation of Akt. Decreased expression ofILK through siRNA action moderately decreased phospho-Akt. In contrast,siRNA-mediated loss of the protein kinases Pctk1, Pftk1, IKKα andMAP4K4/NIK failed to affect the levels of phospho-Akt in the presence orabsence of insulin (FIG. 3), indicating they do not modulate insulinsignaling to Akt2.

Glucose Transporter Expression

We next examined whether the depletion of these protein kinases modulateglucose transporter protein levels in 3T3-L1 adipocytes. Depletion ofILK promotes a significant decrease in GLUT4 mRNA and protein, but notGLUT1 protein (FIG. 4).

These data indicate that inhibition of glucose uptake in adipocytesdepleted of ILK protein is due to a decrease in GLUT4 protein (FIG. 4)and a small decrease in insulin signaling to Akt (FIG. 3). Depletion ofPCTAIRE-1 or PFTAIRE-1 failed to cause detectable changes in GLUT1 orGLUT4-protein levels. In contrast, silencing of IKKα or MAP4K4/NIKexpression promoted a significant increase in cellular GLUT4 protein,but not GLUT1 (FIG. 4), potentially accounting for the enhancement ofdeoxyglucose transport in adipocytes depleted of IKKα or MAP4K4/NIK.

In an attempt to examine whether MAP4K4/NIK also regulates insulinaction on GLUT4 recycling, GFP-GLUT4-myc translocation assays (Jiang etal., Proc. Natl. Acad. Sci. USA, 100:7569-7574, 2003) were performed incontrol or MAP4K4/NIK-depleted cells by siRNA. No effect of MAP4K4/NIKdepletion was detected in insulin-stimulated GFP-GLUT4-myc translocation(data not shown).

Example 3 Characterization of MAP4K4/NIK Modulation of Glucose Transport

MAP4K4/NIK is Unique Among Expressed MAP Kinases in Attenuating GlucoseUptake in 3T3-L1 Adipocytes.

It has been reported that MAP4K4/NIK may mediate the TNFα stimulation ofthe SAPK/JNK pathway, through activation of the TAK1→MKK4/MKK7→JNKcascade (Yao et al., J. Biol. Chem., 274:2118-2125, 1999). Large amountsof TNFα are secreted by adipocytes and macrophages within adipose tissueof obese animals (Wellen and Hotamisligil, J. Clin. Invest., 112:1785-1788, 2003), and this factor a potent negative regulator ofadipogenesis and GLUT4 expression (Zhang et al., Mol Endocrinol.,10:1457-1466, 1996; Stephens et al., J. Biol. Chem., 272:97-976, 1997).TNFα has also been implicated in mediating insulin resistance(Hotamisligil et al., J. Clin. Invest., 95:2409-2415, 1995). Thus, weinvestigated whether the depletion of other members of the MAPK family,including MAP3K7/TAK1, MAP2K4/MKK4, MAP2K7/MKK7, MAPK8A/JNK1 orMAPK9/JNK2 could also enhance glucose transport in adipocytes.

While attenuation of MAP4K4/NIK expression markedly enhancedinsulin-induced glucose uptake, depletion of MAP3K7/TAK1, MAP2K4/MKK4,MAP2K7/MKK7, MAPK8A/JNK1 or MAPK9/JNK2 failed to do so (FIG. 5).Furthermore, FIG. 5 reveals that the effect of MAP4K4/NIK silencing ondeoxyglucose uptake is remarkably specific, since electroporation ofcultured adipocytes with siRNA pools directed against each of the other22 MAPK family members expressed in adipocytes did not enhance insulinsignaling to deoxyglucose transport. In addition, loss of MAP4K4/NIK in3T3-L1 adipocytes had no effect on the ability of TNFα to inducephosphorylation of MAPK8A/JNK1 and MAPK9/JNK2 (data not shown). Takentogether, these results suggest that the enhancement ofinsulin-stimulated deoxyglucose transport observed in cells depleted ofMAP4K4/NIK is not due to disruption of the TNFα→JNK cascade.

MAP4K4/NIK Attenuates Triglyceride Content, PPARγ and C/EBPα Expressionin 3T3-L1 Adipocytes.

We tested whether MAP4K4/NIK regulates adipocyte differentiation.MAP4K4/NIK depletion in 3T3-L1 cells at 4 days after initiation ofdifferentiation indeed enhanced triglyceride content in the cellsmeasured several days later. Cellular triglyceride content wasdetermined spectrophotometrically using a triglyceride determination kit(Sigma). Cells were rinsed and scraped in PBS. Cell suspensions weresonicated and the triglyceride was measured. MAP4K4/NIK depletion in3T3-L1 cells at 4 days after initiation of differentiation indeedenhanced triglyceride content in the cells measured several days later(FIG. 6).

Furthermore, Western blots revealed increased expression of theadipogenic transcription factors, C/EBPβ, C/EBPα and PPARγ uponMAP4K4/NIK gene silencing in 3T3-L1 cells. In contrast, no effect onexpression of SREBP-1, TBP or the structural nuclear protein Lamin A/C,was observed in these same cells. Taken together, these results suggestthat MAP4K4/NIK is unique among the MAP kinases in acting as anendogenous negative regulator of C/EBPβ, C/EBPα and PPARγ expression andadipogenesis in 3T3-L1 cells.

TNFα Treatment and Depletion of PPARγ Enhances MAP4K4/NIK Expression inCultured Adipocytes.

Further analysis of mRNA levels of MAP4K41NIK, PPARγ and C/EBPα duringthe course of 3T3-L1 cell differentiation revealed an inverserelationship between decreasing expression of MAP4K4/NIK and increasingexpression of PPARγ and C/EBPα (FIGS. 7A and 7B). We then tested whetherthe increased PPARγ expression that occurs during adipogenesis maymediate this decrease in MAP4K4/NIK expression. The expression level ofMAP4K4/NIK was examined in fully differentiated 3T3-L1 adipocytesdepleted of PPARγ. As depicted in FIG. 8, a highly significant, 2-foldincrease in MAP4K4/NIK mRNA level was observed in cultured adipocytesupon attenuation of PPARγ expression with RNAi. Thus, PPARγ acts toinhibit the expression of an inhibitor of adipogenesis, MAP4K4/NIK,while MAP4K4/NIK acts to inhibit the expression of a major promoter ofadipogenesis, PPARγ.

In addition to a major role in driving adipogenesis, PPARγ appears tofunction in mature 3T3-L1 adipocytes by maintaining the expression ofgenes that confer the characteristics of fully differentiated adipocytes(Morrison and Farmer, J. Nutr. 130:31 16S-3121 S, 2000; Tamori et al.,Diabetes 51:2045-2055, 2002). Consistent with these previousobservations, attenuation of PPARγ expression by siRNA reduced theabundance of mRNA encoding C/EBPα, GLUT4, PEPCK, aP2, ACS and FAS inmature 3T3-L1 adipocytes (data not shown). Surprisingly, this effect ofPPARγ depletion to decrease the abundance of these genes was markedlyattenuated when MAP4K4/NIK was also depleted in mature 3T3-L1 adipocytes(data not shown). Together, these data imply that in response to theloss of PPARγ in fully differentiated adipocytes, increased MAP4K4/NIKprotein kinase promotes the decay in expression of genes that confer thecharacteristics of mature adipocytes. These results also indicate thatthe maintenance of PPARγ-responsive genes in mature adipocytes isachieved in part through the suppression of MAP4K4/NIK expression byPPARγ.

Finally, we tested whether TNFα, a known negative regulator ofadipogenesis and GLUT4 expression, modulates MAP4K4/NIK. Under theconditions of the present experiments, TNFα treatment of 3T3-L1adipocytes for 24 hours markedly decreased expression of PPARγ andGLUT4, as expected (FIG. 9B). Remarkably, treatment of 3T3-L1 adipocyteswith TNFα for 24 hours caused a 3-fold increase in MAP4K4/NIK mRNAlevels (FIG. 9A). Depletion of MAP4K4/NIK prior to incubation of cellswith TNFα raises the levels of GLUT4 and PPARγ mRNA, and prevents fullinhibition of gene expression by TNFα. Thus, these data are consistentwith the hypothesis that the increased levels of MAP4K4/NIK that appearin response to TNFα treatment of adipocytes contribute to theattenuation of both adipogenesis and expression of GLUT4 mediated byTNFα. These data indicate that TNFα acts to enhance MAP4K4 expressionand to independently suppress PPARγ expression.

Example 4 Evaluating Agents in an Animal Model

Agents that modulate expression or activity of a glucosetransport-related polypeptide or nucleic acid encoding the polypeptidein vitro are further tested in vivo in animal models. For example,scrambled siRNA or siRNA that target one or more genes listed in Table 1are administered to ob/ob mice using hydrodynamic injection aspreviously described (McCaffrey, Nature, 418:38-39, 2002; see also U.S.Pat. Pub. 20030153519). Ob/ob mice can be obtained from JacksonLaboratories (Strain Name: B6.V-Lep^(ob)/J). At various time pointsafter administration of the siRNA, mRNA levels for the target(s) fromTable 1 are measured. Additionally, the siRNA can be labeled and trackedusing methods known in the art. Levels of glucose, glucose tolerance,and plasma insulin can also be monitored to determine whether the siRNAhas a beneficial effect on glucose metabolism, relative to control,i.e., whether the siRNA causes a reduction in hyperglycemia or plasmainsulin levels.

In one embodiment, siRNAs that target Map4K4 are designed and generated.Briefly, fragments of a particular length (e.g., 23 nucleotides) withinthe Map4K4 gene sequence are identified, e.g., as described in U.S. Pat.Pub. No. 20040198682. Fragments containing 40-60% GC content and weakerinternal fold structure (as determined by in silico analysis) arepreferred. Fragments containing strong hairpins and runs of three ormore Cs or Gs are avoided. Four or five target fragments are selectedand synthesized as siRNA duplexes and screened in vitro to identify themost active siRNAs.

Next, the selected Map4K4 siRNA are tested in ob/ob mice in vivo. Toperform hydrodynamic injection, each ob/ob mouse is administered 40micrograms of the selected Map4K4 siRNA in 1.8 mL of PBS. The siRNA/PBSsolution is injected through the tail vein in 4-5 seconds. Levels ofMap4K4 expression are determined by examining Map4K4 RNA and/or proteinlevels in tissues 24 hours, 48 hours, 72 hours, or 4 days afterinjection. Plasma glucose levels in each animal at 1-3 days followingtreatment are also measured and compared to controls (e.g., glucoselevels prior to siRNA treatment and glucose levels in animals treatedwith PBS and scrambled siRNA). Map4K4 siRNA that reduce hyperglycemiacan be useful in treating glucose transport-related disorders such asdiabetes and obesity.

OTHER EMBODIMENTS

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method for identifying a candidate agent that modulates expressionor activity of a glucose transport-related polypeptide, the methodcomprising (a) providing a sample comprising a glucose-transport relatedpolypeptide or a nucleic acid encoding the polypeptide, wherein theglucose-transport related polypeptide is a gene product of a gene inTable 1 or Table 2; (b) contacting the sample with a test compound; (c)evaluating expression or activity of the glucose transport-relatedpolypeptide in the sample; and (d) comparing the expression or activityof the glucose transport-related polypeptide of (c) to expression oractivity of the glucose transport-related polypeptide in a controlsample lacking the test compound, wherein a change in glucosetransport-related polypeptide expression or activity indicates that thetest compound is a candidate agent that can modulate the expression oractivity of the glucose transport-related polypeptide.
 2. The method ofclaim 1, wherein the glucose transport-related polypeptide is a geneproduct of a gene in Table
 1. 3. The method of claim 1, wherein theglucose transport-related polypeptide is a gene product of a gene inTable
 2. 4. The method of claim 1, wherein the sample is a cell.
 5. Themethod of claim 4, wherein the cell is an adipocyte.
 6. The method ofclaim 1, wherein the sample is a cell-free sample.
 7. The method ofclaim 1, wherein evaluating comprises performing a cell-free assay. 8.The method of claim 1, wherein evaluating comprises determining whetherglucose transport is modulated in the presence of the test compound. 9.The method of claim 1, wherein the glucose transport-related polypeptideis a human glucose transport-related polypeptide.
 10. The method ofclaim 1, wherein evaluating comprises determining glucose uptake. 11.The method of claim 1, wherein the test compound is selected from thegroup consisting of a polynucleotide, a polypeptide, a small non-nucleicacid organic molecule, a small inorganic molecule, and an antibody. 12.The method of claim 1, wherein the test compound is selected from thegroup consisting of an antisense oligonucleotide, an inhibitory RNA, anda ribozyme.
 13. The method of claim 8, wherein modulation of glucosetransport is evaluated using an antibody.
 14. The method of claim 8,wherein glucose transport is increased in the presence of the testcompound.
 15. The method of claim 8, wherein glucose transport isdecreased in the presence of the test compound.
 16. The method of claim1, wherein the glucose transport-related polypeptide is a kinase. 17.The method of claim 16, wherein the evaluating comprises determiningphosphorylation of a substrate by the kinase.
 18. A method formodulating glucose transport in a cell, the method comprising: providinga cell; contacting the cell with an agent that modulates expression oractivity of a glucose transport-related polypeptide, thereby modulatingglucose transport in the cell.
 19. The method of claim 18, wherein theagent that modulates expression or activity of a glucosetransport-related polypeptide is identified by the method of claim 1.20. The method of claim 18, wherein the agent modulates the expressionor activity of a gene product of a gene in Table 1 or Table
 2. 21. Themethod of claim 20, wherein the agent decreases expression or activityof a gene product of a gene in Table
 1. 22. The method of claim 20,wherein the agent increases expression or activity of a gene product ofa gene in Table
 2. 23. The method of claim 18, wherein the agent isselected from the group consisting of a polynucleotide, a polypeptide, asmall non-nucleic acid organic molecule, a small inorganic molecule, andan antibody.
 24. The method of claim 23, wherein the agent is a smallinhibitory RNA.
 25. The method of claim 23, wherein the agent isselected from the group consisting of an antisense oligonucleotide, aninhibitory RNA, and a ribozyme.
 26. The method of claim 18, furthercomprising contacting the cell with a second agent that modulatesexpression or activity of a glucose transport-related polypeptide. 27.The method of claim 18, wherein the cell is contacted in vitro.
 28. Themethod of claim 18, wherein the cell is contacted in vivo.
 29. A methodfor increasing insulin-stimulated glucose uptake in a subject, themethod comprising: administering to the subject an agent that decreasesexpression or activity of a gene product of a gene in Table 1 in anamount sufficient to modulate glucose metabolism in a cell of thesubject, thereby increasing insulin-stimulated glucose uptake in thesubject.
 30. The method of claim 29, wherein the subject is at risk foror suffering from a disorder or condition related to glucose metabolism.31. The method of claim 30, wherein the disorder or condition is type Idiabetes, type II diabetes, or obesity.
 32. A method for modulatingglucose metabolism in a subject, the method comprising: administering tothe subject an agent that increases expression or activity of a geneproduct of a gene in Table 2 in an amount sufficient to modulate glucosemetabolism in a cell of the subject, thereby modulating glucosemetabolism in the subject.
 33. The method of claim 32, wherein thesubject is at risk for or suffering from a disorder or condition relatedto glucose metabolism.
 34. The method of claim 33, wherein the disorderor condition is type I diabetes, type II diabetes, or obesity.
 35. Acomposition comprising a nucleic acid encoding an inhibitory RNA thattargets an RNA encoded by a gene of Table
 1. 36. The composition ofclaim 35, wherein the inhibitory RNA is a small inhibitory RNA.
 37. Acomposition comprising an antisense nucleic acid that inhibits thefunction of a gene product of a gene of Table 1.